The unremitting isolation of bacterial pathogens exhibiting resistance to multiple antibiotics demands the development of novel therapeutic strategies. This entails not only identifying resistance determinants but also understanding the environmental cues that regulate expression of such determinants. The Salmonella PmrA/PmrB two-component system is required for resistance to the peptide antibiotic polymyxin B and to several antimicrobial proteins from human polymorphonuclear leukocytes. The regulatory protein PmrA governs expression of proteins mediating modifications of the lipopolysaccharide (LPS) with 4-aminoarabinose, which confers polymyxin resistance, and with phosphoethanolamine, the significance of which has remained unknown. We have established that: (1) the PmrB protein is a sensor that responds to extracellular levels of Fe 3+ by activating the PmrA protein, (2) low Mg2+ can also activate the PmrA protein in a process mediated by the PmrD protein, and (3) mutants defective in the pmrA or pmrB genes are hypersensitive to killing by Fe3+. This proposal describes experiments aimed at understanding the cascade of events by which multiple environmental cues activate the PmrA/PmrB two-component system to promote resistance to different compounds. We will examine the molecular mechanism by which low pH activates the PmrA protein; investigate how the PmrD protein can activate the PmrA protein at a posttranscriptional level; identify the PmrA-regulated determinants mediating resistance to polymyxin and to Fe3+; and define the physiological role of PmrA-controlled phosphoethanolamine modification of the LPS. An accomplishment of these goals will uncover the molecular bases for bacterial signal transduction and antimicrobial peptide resistance. Moreover, as a variety of peptides and proteins are currently being developed as novel antimicrobial agents, the proposed experiments may help in our understanding of how these compounds exert their microbicidal properties. [unreadable] [unreadable]